7-(3&#39;-methyl-4&#39;-furazanacetamido)-cephalosporanic acid and 6-(3&#39;-methyl-4&#39;-furazanacetamido) penicillanic acid and related salts and derivatives



3,322,751 '7 (3 METHYL-4' lFURAZANAClETAMlDW-CEPH- ALOSPORANIC ACID AND6 (3' METHYL 4'- FURAZANACETAMEDOWENECILLANIC A C l D AND RELATED SALTSAND DERIVATIVES Leonard Bruce Crast, 31a, North Syracuse, N.Y., assignorto Bristol-Myers Company, New York, N.Y., a corporation of Delaware NoDrawing. Filed Mar. 30, 1966, Ser. No. 538,611 Claims. (Cl. Mil-239.1)

This invention relates to new synthetic compounds of value asantibacterial agents, as nutritional supplements in animal feeds, asagents for the treatment of mastitis in cattle, and as therapeuticagents in poultry and animals, including man, in the treatment ofinfectious diseases caused by Gram-positive and Gram-negative bacteriaand especially salmonella and, more particularly, relates to 7(3'-m=ethy1-4-furazanacetamido)-cephalosporanic acid and6-(3-methyl-4-furazanacetamido)-penicil1anic acid and related salts andderivatives.

Antibacterial agents of the penicillin class have proven highlyeffective in the therapy of infections due to either Gram-positive orGram-negative bacteria but few are effective against both, very few areeffective in concentrations below 1.0 meg/ml. and none are veryeffective in practical use against infections caused by Salmonella, e.g.S. enteritidis. It was the object of the present invention to providenovel compounds effective against both Gram-positive and Gram-negativebacteria including the resistant strains. It was a further object of thepresent invention to provide penicillins and cephalosporins activeagainst Gram-positive and Gram-negative bacteria which are alsoefficiently absorbed upon oral administration to man and animals.

The objects of the present invention have been achieved by theprovision, according to the present invention, of the acids of theformula and nontoxic, pharmaceutically acceptable salts thereof.

The objects of the present invention have also been achieved by theprovision, according to the present invention, of the compounds of theformula A is hydrogen, hydroxyl, (lower) alkanoyloxy containing 2-8carbon atoms, e.g. acetoxy, propionoyloxy, butanoyloxy, pentanoyloxy,etc., benzoyloxy, a quaternary ammonium radical, e.g. pyridinium,quinolinium, picolinium, lutidinium, or, when taken together with M, amonovalent carbon-oxygen bond; and

M is hydrogen, a pharmaceutically acceptable nontoxic cation, an anioniccharge when A is a quaternary ammonium radical, or, when taken togetherwith A, a monovalent carbon-oxygen bond.

For clarity, we have illustrated below the formulae of the compoundswhen, in Formula I, A is a quaternary ammonium radical (pyridiniurn) andM is an anionic 3,322,751 Patented May 30, 1967 charge and when A and Mtogether are a monovalent carbon-oxygen bond.

The preferred embodiments of the present invention are the free acidsand salts thereof of which the free acids have the formulae Thepharmaceutically-acceptable, nontoxic cations include metallic cationssuch as sodium, potassium, calcium and aluminum and organic aminecations such as trialkylamines, e.g. triethylamine, pnocaine,dibenzylamine, N- lbenzyl-B-phenethylamine, l-ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine,N,N-'bis-dehydroabietylethylenediamine, N (lower)alkylpiperidines, e.g.N-ethylpiperidine, and other amines which have been used to form saltswith benzylpenicillin.

The products of the present invention are prepared by the reaction of6-aminopenicillanic acid or a compound of the formula wherein A and Mare described above (preferably in the form of a neutral salt such asthe sodium salt or the triethyl'amine salt, i.e. when A is hydrogen,hydroxy, (lowerlalkanoyloxy or benzoyloxy) with an active ester, etg.2,4-dinitr0phenyl ester, p-nitrophenyl ester or N-hydroxysuccinimideester, of an acid having the formula or with its functional equivalentas an acylating agent for a primary amino group. Such equivalentsinclude the corresponding carboxylic chlorides, bromides, acidanhydrides, including mixed anhydrides and particularly the mixedanhydrides prepared from stronger acids such as the lower aliphaticmonoesters of carbonic acid, of alkyl and aryl sulfonic acids and ofmore hindered acids such as diphenylacetic acid. In addition, an acidazide or an active ester or thioester (e.g. with p-nitro phenol,2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the freeacid itself may be coupled with 7-aminoacephalosporanic acid after firstreacting said free acid with N. N-dimethylchloroformininium chloride[cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/ 6,360* (1965)] or by the use of enzymes or of an N,N-carbonyldiimidazoleor an N,N'-carbonyditriazole [of South African patent specification63/2684] of a carbodiimide reagent [especiallyN,N-dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide orN-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide; cf. Sheehan and Hess,J. Amer. Chem. Soc., 77, 1067 (1955)], or of alkynyla-mine reagent [cfR. Buijle and H. G. Viehe, Agnew. Chem. International Edition, 3, 582(1964)], or of a ketenirnine reagent [cf. C. L. Stevens and M. E. Monk,J. Amer. Chem. Soc., 80, 4065 (1958)] or of an isoxazolium salt reagent[of R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc, =83,1010 (1961)]. Another equivalent of the acid chloride is a correspondingazolide, i.e. an amide of the corresponding acid whose amide nitrogen isa member of a quasiaromatic five-membered ring containing at least twonitrogen atoms, i.e. imidazole, pyrazole, the triazoles, benzimidazole,benzotriazole and their substituted derivatives. As an example of thegeneral method for the preparation of an azolide,N,N-carbonyldiimidazole is reacted with a carboxylic acid in equirnolarproportions at room temperature in tetrahydrofuran, chloroform,dimethylforrnamide or a similar inert solvent to form the carboxylicacid imidazolide in practically quantitative yield with liberation ofcarbon dioxide and one mole of imidazole. Dicarboxylic acids yielddiimidazolides. The by-product, imidazole, precipitates and may beseparated and the imidazolide isolated, but this is not essential. Themethods for carrying out these reactions to produce a cephalosporin andthe methods used to isolate the cephalosporin so produced are well-knownin the art (cf. US. Patents Nos. 3,079,314, 3,117,126 and 3,129,224 andBritish Patents Nos. 932,644, 957,570 and 959,054).

- The compounds of Formula VI used in the present invention include7-aminoceph-alosporanic acid and derivatives of 7-aminocephalosponanicacid. 7-a-minocephalosporanic acid is prepared by hydrolysis ofcephalosporin C and has the formula Acid hydrolysis of cephalosporin Cto produce 7- aminocephalosporanic acid results in the coproduction ofthe lactone, 3-hydroxymethyl-7-aminodecephalosporanic acid lactone,formed by the further hydrolysis of the acetoxy group and subsequentinternal esterification. The lactone has the formula 4 Enzymatichydrolysis of the acetoxy group of 7-aminocephalosporanic acid resultsin the formation of 3-hydroxymethyl-7-aminodecephalosporanic acid havingthe formula and such compound may be re-esterified with benzoic acid ora lower alkanoi-c acid, e.g. acetic acid, propionic acid and the like toform other esters. Preferably, reesterification is carried out on a3-hydroxymethyl-7-(othydroxy-thienylacetamido)decephalosporanic acidwhich is obtained by enzymatic hydrolysis of a7-(a-hydroxythienylacetamido)cephalosporanic acid.

Treatment of cephalosporin C with a tertiary amine, e.g. pyridine,lutidines, picolines and the like, followed by acid hydrolysis producesa nucleus which, in the case of pyridine, has the formula and has beengiven the name 3-pyridiniummethyl-7- aminodecephalosporanic acid innersalt.

The foregoing nuclei and the preparation thereof are known in the artand are described for example in US. Patent No. 3,117,126 and BritishPatents Nos. 932,644, 957,570 and 959,054.

3-rnethyl-7-aminodecephalosporanic acid having the formula is producedby catalytic reduction of cephalosporin C followed by hydrolytic removalof the S-aminoadipoyl side chain as described in US. Patent No.3,129,224.

STARTING MATERIALS The ring system having the structure 011 l l N5 2N isnamed 1,2,5-oxadiazine or furazan with the atoms numbered as indicated.

The 3-methyl-4-furazanacetic acid used in the present invention isprepared, for example, in the manner set forth in Example 1 below from3methyl-4-furazancarboxylic acid which in turn is prepared according tothe literature, e.g., Berichte, 28, 70, 71 (1895), by oxidation ofcommercially available 3,4-dimethyl-1,2,5-oxadiazine.

The following examples will serve to illustrate this invention withoutlimiting it thereto. All melting points are uncorrected and alltemperatures are given in degrees centigrade.

Example 1 N A100 C.

(I) 3-methyl-4-furazancarb0xylic acid.Via Berichte 28, 70, 71 (1895).

(II) 3-methyl-4-furazancarbonyl chloride-A mixture of 50 g. (0.39 mole)of 3-methyl-4-furazancarboxylic acid, 150 ml. of thionyl chloride and 1ml. of dirnethylforrnamide (DMF) were heated at reflux for four hours.The product distilled at 70 C./0.25 mm. Hg to give 57 g. (100%) of3-methyl-4-furazancarbonyl chloride.

(III) 3-mezhyl-4-furazanacetic acid.To a stirred and cooled solution ofapproximately 0.250.3 mole of diazomethane in 500 ml. of ether wasadded, dropwise 14.65 g. (0.1 mole) of 3-methyl-4-furazancarbonylchloride in 100 ml. of dry ether over a ten minute period. The ice bathwas then removed and stirring continued for one hour. The ether was thenremoved in vacuo to leave an oil which was not purified further.

The oil was then heated to 182 C. in 7 0 ml. of benzyl alcohol and 70ml. N,N-dimethylaniline for one hour at which time nitrogen evolutionceased. The mixture was then cooled to 20 C. and diluted with 500 ml. ofether. Next, three 300 ml. 6 N HCl extracts were taken and discarded.After one 300 ml. water wash the ether solution was evaporated to an oilat 22 C. under reduced pressure. This oil was then heated 16 hours in 80ml. cone. hydrochloric acid and 80 ml. of glacial acetic acid on thesteam bath. The mixture was cooled and the pH adjusted to 8 with 20%NaOH (aqueous). Three 600 ml. ether extracts were taken and discarded.The aqueous phase was cooled and acidified to pH 2 with cone. HCl andthen saturated with salt. Three 300 ml. ether extracts were then taken,combined and evaporated to an oil. The oil was dried by adding severalportions of benzene and removing same under reduced pressure. The oilthen crystallized upon cooling and was recrystallized from toluene togive 6.6 g., M.P. 83-84 C. of 3-methyl-4-furazanacetic acid.

Analysis.-Calcd for C H N O C, 42.25; H, 4.23; N, 19.98. Found: C,42.81; H, 4.38; N, 19.79.

(IV) Potassium 6-(3 methyl 4 furazanacetamido)- penicillanate.--To 0.590g. (0.00415 mole) of 3-methyl- 4-furazanacetic acid was added 3 ml. ofthionyl chloride and the mixture heated on the steam bath for twentyminutes. The excess SOC1 was then removed under reduced pressure and thecrude acid chloride was dissolved in 5 ml.

of acetone and added dropwise to a previously prepared, vigorouslystirred, solution of 1.08 g. (0.005 mole) of 6-aminopenicillanic acid,1.68 g. (0.020 mole) of sodium bicarbonate in 10 ml.- of water and 5 ml.of acetone at 5 C. The mixture was stirred for one hour with theice-salt bath removed and then diluted with 10 ml. of water and theacetone removed under reduced pressure at 22 C. The resulting aqueoussolution was extracted with one 30 ml. portion of ether and then layeredwith 20 ml. of ether and cooled and acidified to pH 2 with 40% H PO Theether extract was then washed with three 20 ml. portions of water anddried 10 minutes over magnesium sulfate. The ether solution was thenfiltered and the MgSO washed with three 5 ml. portions of ether. Thecombined washes and filtrate were treated with 2.5 ml. of 50% KEH(potassium 2-ethylhexanoate in n-butanol) and scratched. There wasobtained 500 mg. of acetone washed and vacuum dried potassium6-(3-methyl-4- furazanacetarnido)-penicillanate decomposing at 202- 203C. IR and NMR analysis were consistent with the desired structure.

Analysis.Calcd for C H N O S-K: C, 41.27; H, 4.00; N, 14.81. Found: 1 C,41.27; N, 14.54; H, 3.91.

This compound exhibited M.I.C.s versus S. typhosa and K]. pneumoniae ofabout 3.1 mcg./ml. and versus S. enteritidis of about 0.8 mcg./ml.

Example 2.S0dium 7-(3-mezhyl-4-furazanacetamid0)- cephalosporarzate To2.84 g. (0.020 mole) of 3-methyl-4-furazanacetic acid was added 10 m1.of thionyl chloride and the mixture heated 30 minutes on the steam bath.The excess SOCl Was then removed under reduced pressure at 22 C. and theresidual oil dissolved in 20 ml. of methylene chloride and addeddropwise to a previously prepared solution of 5.44 g. (0.020 mole) of7-aminocephalosporanic acid, 5.6 ml. (0.040 mole) of triethylamine and60 ml. of CH Cl at 0 C., with stirring over a 15 minute period. Thecooling bath was then removed and stirring continued for one hour. Theresulting solutiong' ijwas concentrated to an oil under reduced pressureat 22 C. and then 75 ml. of 2% NaHCO solution (aqueous) and ml. of etherwere added. After shaking, the aqueous phase was separated and layeredwith 75 ml. of ethyl acetate. With cooling and stirring, 40% H PO wasadded until pH 2 was obtained. The ethyl acetate extract was thenseparated and washed three times with 50 ml. portions of water and twicewith 50 ml. portions of saturated salt solution. The ethyl acetatesolution was then dried 10 minutes over MgSO filtered and the MgSOwashed with three 20 ml. portions of ethyl acetate and combined with thefiltrate. Next, 7 ml. (0.020 mole) of sodium 2-ethylhexanoate innbutanol Was added and with scratching the product began to crystallize.After 30 minutes the product was filtered off, washed with ethyl acetateand air dried. The yield was 3.8 g. The product was found to be solublein acetone but would crystallize to a different crystalline form whenthe flask was scratch-ed. By this method the product was recrystallizedto the acetone-insoluble form to yield 1.8 g. sodium 7 (3 methyl 4furazanacetamido) cephalosporanate decomposing at 182 C. The IR and NMRspectra were consistant with the desired structure.

Analysis.Calcd for C15H15N4O7S'NZLI C, 43.06; H, 3.62; N, 13.40. Found:2 C, 43.77; H, 3.67; N, 13.17.

This compound exhibited M.I.C.s of about 0.8 meg/ml. versus thebenzylpenicillin-resistant Staph. aureux BX- 1633-2 and about 1.6meg/ml. versus S. enteritidis and of about 6.2 mcg./ml. versus S.ryphosa and of about 6.2- 12.5 meg/ml. versus Kl. pneumoniae and washighly active versus Shig. sonnei.

Water as determined by the Karl Fischer method.

'7 Example 3 3-pyridiniummethyl-7-aminodecephalosporanic acid inner salt(1.5 g.) is shaken with methylene chloride at ;room temperature untilthe mixture becomes homogeneous and this solution is used in place ofthe 74amino- -cephalosporanic acid solution in the procedure of Ex-:ample 2 to prepare 3-pyridiniummethyl-7-[a-(3-methyl-4-furazan)acetamido]decephalosporanic acid inner salt. This product issensitive to light so that it is advisable to protect it from light asmuch 13S practical during its manufacture and subsequent processing andpackaging.

Example 4 3rnethyl-4-furazan-acetic acid (0.002 mole) and 2,4-dinitrophenol (0.002 mole) are dissolved in dry dioxane (10 ml.) and thesolution is cooled in an ice bath. N,N- dicyclohexylcarbodiimide (0.002mole) is added and the solution is shaken well and left at roomtemperature for 45 minutes. The precipitated urea is removed byfiltration and washed with ethyl acetate (25 ml.). The filtrate andwashings are combined and concentrated in vacuo at room temperature toleave as the residue the desired 2,4-dinitrophenyl3-methyl-4-furazan-acetate.

3-pyridiniummethyl-7-aminodecephalosporanic acid inner salt (0.002 mole)is shaken with methylene chloride at room temperature until the mixtureis homogeneous. The mixture is cooled in an ice bath and2,4-dinitrophenyl 3-methyl-4-furazan-acetate (0.002 mole) is added withshaking and the resulting solution left at room temperature untilcompletion of the reaction. The reaction is followed by measuring theintensity of the amide absorption band at 1675 cm? in the infraredspectrum. The mixture is filtered and addition of ether precipitates theproduct, 3 pyridiniummethyl-7-[a-(3-methyl-4-furazan)acetamido]decephalosporanic acid inner salt. The product is dissolved inmethylene chloride, reprecipitated with ether, collected, dried andfound to contain the fl-lactam structure as shown by infrared analysis,to inhibit Staph. aureus at low concentrations and to be highly solublein water.

Example Pyridine (10 ml.) is added with stirring to a mixture of water(50 ml.) and 7-[a-(3-methyl-4-furazan)acetamido1cephalosponanic acid (5g.) to form a solution which is left under nitrogen at about 45 C. fortwelve hours and then extracted four times with 20 ml. methylenechloride. The aqueous phase is concentrated in vacuo at about 30 C. andthen passed through a column containing a strongly basic, anion exchangeresin of the quaternary ammonium type (e.g. Dowex 1) in the acetatecycle. The eluates containing the desired pyridine derivative as judgedpolarimetrically are combined, lyophilized and triturated in methanol togive solid 3-pyridiniummethyl- 7[a-(3-methyl-4-furazan)acetamido]decephalosporanic acid inner salt. Byconcentrating the methanol triturate at 30 C. in vacuo and then pouringthe concentrate so obtained into a large volume of acetone there isprecipitated an additional amount of this product.

Example 6 Substitution of an equal volume of a-picoline, 'y-picoline and2,4-lutidine, respectively, for pyridine in the procedure of Example 5produces 3-a-picoliniummethyl-7-[a-(3-methyl-4-furazan)acetamido]decephalosporanic acid inner salt,

8 3- -picoliniummethyl-7- [a- 3-methyl-4-furanan)acetamido]decephalosporanic acid inner salt, and3-2,4-lutidiniummethyl-7- a- 3-methyl-4-furazan) acet-.-amido]decephalosporanic acid inner salt, respectively.

Example 7 Substitution of an equimolar amount of3-*y-picoliniummethyl-7-aminodecephalosporanic acid inner salt for the3-pyridinium-7-aminodeoephalosporanic acid inner salt in the procedureof Example 4 produces 3-v-picoliniummethyl 7 [oz (3 methyl 4furazan)acetamido]decephalosponanic acid inner salt.

Example 8 The product of Example 2 is dissolved in water and treatedwith acetylesterase obtained from orange peels according to theprocedure of Jansen et al., Arch. Biochem. 15, 415 (1947) at pH 6 for 15hours. The resulting solution is passed through a column containing aweak, anionic ion exchange resin (e.g. Amberlite IR 413) in the acetateform and the column is then eluted with aqueous acetic acid which hasbeen adjusted to pH 5.5 with pyridine. The eluate is adjusted to pH 8 bythe addition of sodium hydroxide and is then evaporated in vacuo to give3-hydroxymethyl-7-[a-(3-methyl 4 furazan) acetamido]decephalosporanicacid in the form of its sodium salt.

Example 9 When in Example 2 the 7-aminocephalosporanic acid is replacedby an equimolar amount of 3-methyl-7-aminodecephalosporanic acid thereis obatined the sodium salt of 3-methyl-7-[a-(3 methyl 4furazan)acetamido]decephalosporanic acid.

Example 10 3-hydroxymethyl 7 aminodecephalosporanic acid lactone (0.002mole) is shaken with methylene chloride at room temperature until themixture is homogeneous. The mixture is cooled in an ice bath and2,4-dinitrophenyl-3- methyl-4-fi1razan-acetate (0.002 mole) is addedwith shaking and the resulting solution is held at room temperatureuntil completion of the reaction is followed by measurement of theintensity of the amide absorption band at 1675 CHIC-1 in the infraredspectrum. After filtration, the addition of ether to the filtrateprecipitates 3-hydroxy-methyl-7-[a-(3-methyl 4furazan)acetamido]decephalosporanic acid lactone. The product isdissolved in methylene chloride, reprecipitated by adding ether,collected by filtration and dried.

Example 11 7-aminocephalosporanic acid (0.001 mole) and triethylamine(0.004 mole) are shaken in methylene chloride (2 ml.) until the mixtureis homogeneous. This mixture is cooled in an ice bath and2,4-dinitrophenyl-3-methyl-4- furazan-acetate (0.001 mole) dissolved in3 ml. methylene chloride is added with shaking; the resulting solutionis allowed to stand at room temperature for two hours to complete thereaction. The progress of the reaction is followed by measuring theintensity of the amide absorption band at 1675 cm. in the infraredspectrum. The addition of dry ether precipitates the triethylamine saltof 7- a- 3-methyl-4-furazan) -acetamido] cephalospor anic acid.

Example 12 7-aminocephal0sporanic acid (10 g., finely divided) issuspended in boiling ethyl acetate (400 ml.) andS-methyl-4-furazan-acetyl chloride (10 g.) in ethyl acetate (40 ml.) isadded. The mixture is boiled under reflux for one hour, cooled andfiltered. Aniline (10 ml.) is added and after one hour the mixture isextracted four times with 200 ml. portions of 3% aqueous NaHCO and thecombined alkaline, aqueous extracts are extracted three times with ethylacetate (200 ml. portions), dis- 9 w carding the ethyl acetate extract.The aqueous solution is benzoyloxy, a quaternary ammonium radical of theacidified to pH 1.2 and the product, 7-[ot-(3-methy1-4- formula furazan)acetamido]cephalosporanic acid, is twice ex- 2 tracted into ethylacetate (300 ml.) portions. The com- 69 bined ethyl acetate extracts arewashed with water 5 (4 x 100 ml.), dried over anhydrous MgSO filtered toremove the drying agent and concentrated in vacuo' at room temperatureto precipitate the product, which is re- Wherem each o R and R ishydrogen or methyl or, crystallized, if desired, from aqueous acetone orethanol. when taken together Wlth a momma-lent carbon- While in theforegoing specification various embodioxygen bond; and ments of thisinvention have been set forth in specific M 18 hYdrogen pllarmaceutlcanyaFceptable nontoxm detail and elaborated for the punpose ofillustration, it i an 'f charge when A quaterflary will be apparent tothose skilled in the art that this invenmonmm radlcal when takentogether Wlth a monovalent carbon-oxygen bond.

t1on 1s susceptible to other embodiments and that many 15 3. A compoundof claim 2 having the formula of the details can be varied widelywithout departing from the basic concept and the spirit and scope of theinvention. f

I claim: CHa-CCCHz-CNHCHCH 0H, 1. The acid of the formula P i 20 OCHa-CC-CHTC-NHCH-CH G-GHQ C OM II II I I wherein M 1s a nontoxic,pharmaceutlcally acceptable N\ /N O=GN-CHCOOH cation 0 4. A compound ofclaim 2 having the formula 5 and nontoxic, pharmaceutically acceptablesalts thereof. CHPC 2. A compound of the formula II II I I 69 N N O=CNCCHz S O I z c113oc-oH2i :-NH-oH-ofi oHl Q Q l wherein R and R are eachhydrogen or methyl.

\ 5. The compound of claim 4 wherein R and R are C each hydrogen.

I 0 0 on i No references clted.

wherein A is hydrogen, hydroxyl, (lower)-alkanoyloxy, NICHOLAS RIZZQ,Primary Examiner.

UNITED STATES PATENT OFFICE CERT'IFICATE OF CORRECTION Patent No 3 ,322,751 May 30 1967 Leonard Bruce Crast, Jr.

It is hereby certified that error appears in the above numbered patentrequiring correction and that the said Letters Patent should read ascorrected below.

Column 1, lines 50 to S6, formula I should appear as shown below insteadof as in the patent:

O CH C CCH -!I -NH-CHCH CH N l! O=( ?I J C I-CH A column 3 line 15 for"N,Nrcarbonyditriazole" read N,N carbonylditriazole column 5, lines 16to 19, the formula should appear as shown below instead of as in thepatent:

H3C CH2-CO2CH2 HOAc, HCl

column 8, line 31, for "obatined" read obtained column 10, line 35,strike out "No references cited." and insert the following:

References Cited by the Applicant Signed and sealed this 30th day (SEAL)Attest:

EDWARD M.FLETCHER,JR.

Attesting Officer UNITED STATES PATENTS 6/1960 Doyle et a1. 9/1960 Doyleet a1 5/1961 Doyle et a1 8/1961 Doyle et a1 3/1965 Hobbs et a1.

FOREIGN PATENTS of July 1968.

EDWARD Ja BRENNER Commissioner of Patents

1. THE ACID OF THE FORMULA
 2. A COMPOUND OF THE FORMULA